BUSCADOR RECOLECTA

S1 Fig. The crm1-T539C mutation does not substantially impair meiosis or checkpoint function in the absence of LMB. (A) Sporulation efficiency was examined after 3 days on sporulation plates. Error bars, SD; n = 3. At least 300 cells were counted for each strain. Strains are: DP421 (wild type), DP1717 (crm1-T539C GFP-PCH2), DP422 (zip1Δ), and DP1721 (zip1Δ crm1-T539C GFP-PCH2). (B) Time course analysis of meiotic nuclear divisions. The percentage of cells containing two or more nuclei is represented. Ethanol (Mock) or Leptomycin B (LMB) were added 15 h after meiotic induction (arrow). Error bars: SD; n = 3. At least 300 cells were scored for each strain at every time point. Strains are: DP1620 (ZIP1 CRM1 GFP-PCH2), DP1717 (ZIP1 crm1-T539C GFP-PCH2), DP1621 (zip1Δ CRM1 GFP-PCH2) and DP1721 (zip1Δ crm1-T539C GFP-PCH2), Peer reviewed

(A-B) Immunofluorescence of spread meiotic chromosomes at pachytene stained with anti-GFP antibodies (to detect GFP-Pch2; green), anti-Zip1 antibodies (red) and DAPI (blue). Representative nuclei are shown. In both, (A) and (B), cultures were mock-treated, or treated with 500 ng/ml LMB 15 h after meiotic induction. In (B), Auxin (500μM) was also added 12 h after meiotic induction to degrade Orc1. Spreads were prepared at 19 h. Arrows point to the rDNA region (nlo) and Polycomplex (PC). Scale bar, 2 μm. The strain in (A) is: DP1717 (crm1-T539C GFP-PCH2). The strain in (B) is: DP1885 (orc1-3mAID crm1-T539C GFP-PCH2). (C) Percentage of nuclei containing polycomplexes in the experiments shown in (A and B). Between 10 to 20 nuclei were counted for each strain and condition. (D) Quantification of the different patterns of Pch2 localization in the experiment presented in Fig 3A and 3B. Representative cells are shown., Peer reviewed

(A) Schematic representation of the S. cerevisiae Pch2 protein. The position and sequence of the three putative NESs predicted by LocNES in the non-catalytic N-terminal domain of Pch2 are depicted, as well as the corresponding mutants generated. The images show representative zip1Δ pch2Δ cells transformed with centromeric plasmids expressing wild-type GFP-PCH2 or the different mutated versions of the predicted NESs, as indicated. Note that only the mutation of the 205–214 region (boxed) leads to Pch2 accumulation in the nucleus. Images were taken 15 h after meiotic induction. The strain is DP1405 (zip1Δ pch2Δ) transformed with the centromeric plasmids pSS393 (GFP-PCH2), pSS448 (GFP-pch2-ntd98-107-6A), pSS451 (GFP-pch2-ntd127-136-5A) and pSS459 (GFP- pch2-ntd205-214-4A). (B) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (A). Error bars, SD. (C) AlphaFold model of Pch2 structure. The N-terminal domain of Pch2 is labeled in pink. The positions of the putative NESs analyzed are labeled in yellow (98–107 region), green (127–136) and blue (205–214)., Peer reviewed

ClustalW alignment of the protein sequences of Pch2 orthologs from S. cerevisiae (ScPch2) and human (HsTRIP13). The characteristic AAA+ ATPase features are boxed in blue. The presumed NESs analyzed are boxed in red. The color code for amino acids is the following: AVFPMILW (small + hydrophobic -Y): red. DE (acidic): blue. RK (basic -H): magenta. STYHCNGQ (hydroxyl +sulfhydryl + amine + G): green. Alignment was performed at https://www.ebi.ac.uk/Tools/msa/clustalo/., Peer reviewed

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments., Peer reviewed

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments., Peer reviewed

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments., Peer reviewed

Excel workbook with separate spreadsheets containing numerical data underlying the corresponding figure panels., Peer reviewed

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments., Peer reviewed

Sequences of oligonucleotides used in the construction of strains and plasmids., Peer reviewed